Unlike RNA-TBA (rTBA), iso-rTBA, the 2'-5'-linked RNA-thrombin-binding aptamer, is a functional equivalent of TBA.

An antiparallel, functional RNA G-quadruplex of the 2'-5'-linked thrombin-binding aptamer (iso-rTBA) is reported for the first time. It can inhibit clotting and is remarkably stable to nuclease-degradation, besides having high thermal stability. It is thus, a superior candidate to TBA, rTBA or isoTBA, for further development as an anticoagulant.

Influence of fluorine substitution on the molecular conformation of 3'-deoxy-3'-fluoro-5-methyluridine.

Fluorine substitutions on the furanose ring of nucleosides are known to strongly influence the conformational properties of oligonucleotides. In order to assess the effect of fluorine on the conformation of 3'-deoxy-3'-fluoro-5-methyluridine (T), CHFNO, we studied its stereochemistry in the crystalline state using X-ray crystallography. The compound crystallizes in the chiral orthorhombic space group P222 and contains two symmetry-independent molecules (A and B) in the asymmetric unit.

Methoxymethyl Threofuranosyl Thymidine (4'-MOM-TNA-T) at the T7 Position of the Thrombin-Binding Aptamer Boosts Anticoagulation Activity, Thermal Stability, and Nuclease Resistance.

The synthesis of 4'-methoxymethyl threofuranosyl (4'-MOM-TNA) thymidine and derived oligomers of the G-rich thrombin-binding aptameric (TBA) sequence is reported. The G-quadruplex stability, anticoagulation activity, and the enzymatic stability of these oligomers bearing the 2'-3'-phosphodiester backbone as single substitutions in the loop regions are studied. Amongst all the oligomers, TBA-7T bearing the 4'-MOM-TNA unit at the T7 position formed a quadruplex with the highest thermal stability.

Synthesis Protocols for Simple Uncharged Glycol Carbamate Nucleic Acids.

Glycol carbamate nucleic acid (GCNA) oligomers can be produced from activated carbonate monomers. The synthesized monomers can be very conveniently characterized employing analytical tools like NMR and HR-MS. Moreover, the activated carbonate monomers do not require coupling agents, and hence excess monomers can be recovered at the end of each coupling.

Favorable 2'-substitution in the loop region of a thrombin-binding DNA aptamer.

Simple 2'-OMe-chemical modification in the loop region of the 15mer G-rich DNA sequence GGTTGGTGTGGTTGG is reported. The G-quadruplex structure of this thrombin-binding aptamer (TBA), is stabilized by single modifications (T → 2'-OMe-U), depending on the position of the modification. The structural stability also renders significantly increased inhibition of thrombin-induced fibrin polymerization, a process closely associated with blood-clotting.

Evolution of specific 3'-5'-linkages in RNA in pre-biotic soup: a new hypothesis.

This article reviews the different possibilities towards progression of the formation of DNA/RNA in the chemical world, before life, in enzyme-free conditions. The advent of deoxyribo- and ribopentose-sugars, nucleosides, nucleotides and oligonucleotides in the prebiotic soup is briefly discussed. Further, the formation of early single stranded oligomers, base-pairing possibilities and information transfer based on the stability parameters of the derived duplexes is reviewed.

4'-Epi-DNA: A DNA Mimic Containing 4'-hydroxymethyl-α-l-Xylo-Thymidine with Compact Backbone like RNA.

Synthesis of C4'-epi-DNA containing 3'→ 5″ linkages is reported for the first time. Crystal structure study of the monomer indicated that though the dihedral angle O3'-C3'-C4'-C5″ in this case would be like in RNA, the sugar conformation would remain like that in DNA. The study of the effect of this backbone configuration in DNA with respect to its binding to cDNA and RNA is reported in this note.

Inhibition of miR-21 by 3'/5'-Serinyl-Capped 2'-O-Methyl RNA Interspersed with 2'-O-(2-Amino-3-Methoxypropyl) Uridine Units.

miRNAs are highly conserved class of small ncRNAs whose involvement in human pathophysiologies is extensively investigated. MiR-21 is a well established oncogenic miRNA whose deregulation plays a significant role in onset and progression of cancer. The need of novel approaches to downregulate miR-21 is rapidly expanding.

Efficient Cellular Entry of (r-x-r)-Type Carbamate-Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin.

Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.

Unimolecular antiparallel G-quadruplex folding topology of 2'-5'-isoTBA sequences remains unaltered by loop composition.

A 2'-5'-linked isoTBA 15 mer sequence with (232) loop composition formed stable antiparallel quadruplex structures similar to the SELEX derived 15 mer TBA sequence with (232) loop composition. A parallel versus antiparallel topology of 3'-5'-G-quadruplexes is largely dictated by the loop length, and it is known that the truncated loops favour parallel quadruplexes. In contrast to TBA, systematic reduction of the loop length in isoTBA from (232) to (222), (131) or even (111) did not alter the antiparallel topology of the resulting 14 mer, 13 mer and 11 mer G-rich modified isoTBA-like sequences.

Glycine-Linked Nucleoside-β-Amino Acids: Polyamide Analogues of Nucleic Acids.

3'-5'-Deoxyribose-sugar-phoshate backbone in DNA is completely replaced by 2'-deoxyribonucleoside-based β-amino acids interlinked by glycine to create uncharged polyamide DNA with 3'-5'-directionality. These oligomers as conjugates of α-amino acids and nucleoside-β-amino acids bind strongly and sequence-specifically only to the antiparallel complementary RNA and DNA.

β,γ-Bis-substituted PNA with configurational and conformational switch: preferred binding to cDNA/RNA and cell-uptake studies.

(S,S)- and (R,R)-β,γ-Bis-substituted PNAs were synthesized from the C-2 symmetric vicinal diamine system embedded in 1,4 dihydroxybutane and 1,4-dimethoxybutane scaffolds. (R,R)-β,γ-Bis-methoxymethyl-PNA derived from d-tartaric acid was found to be in the right configuration and conformation to be an excellent mimic of PNA, endowed with superior ability to enter into cells.

Simple molecular engineering of glycol nucleic acid: progression from self-pairing to cross-pairing with cDNA and RNA.

The acyclic chiral nucleic acid analogue, Glycol Nucleic Acid (GNA), displayed exceptional structural simplicity and atom economy while forming self-paired duplexes, using canonical Watson-Crick base pairing. We disclose here that the replacement of phosphodiester linker in GNA with somewhat rigid and shorter carbamate linker in Glycol Carbamate Nucleic Acid (GCNA) backbone allows unprecedented stability to the antiparallel self-paired duplexes. The R-GCNA oligomers were further found to form cross-paired antiparallel duplexes with cDNA and RNA following Watson-Crick base pairing.

Second generation, arginine-rich (R-X'-R)(4)-type cell-penetrating α-ω-α-peptides with constrained, chiral ω-amino acids (X') for enhanced cargo delivery into cells.

The syntheses of novel N-aminoalkyl proline-derived spacers (X') in polycationic (R-X'-R)-motif cell-penetrating α-ω-α-peptides are described as improved molecular transporters and their structural features studied by CD. FACS analysis shows enhanced cellular uptake and confocal microscopy indicates predominantly cytoplasmic localization. The oligomers are efficient at transporting pDNA into cells.

Enhanced splice correction by 3', 5'-serinol and 2'-(ω-O-methylserinol) guarded OMe-RNA/DNA mixmers in cells.

Development of artificial nucleic acids for therapeutic applications warrants that the oligomers be endowed with high specificity, enzymatic stability and with no/reduced off-target effects. The balance between strength of the duplex with target RNA and enzyme stability is therefore the key factor for the designed modification. The chiral serinol derivative combines the attributes of amino- and methoxy- substitution when at 2'- position and at 3'- and 5'- ends, effectively balancing the duplex stability and resistance to hydrolytic enzymes.

Functional isoDNA aptamers: modified thrombin binding aptamers with a 2'-5'-linked sugar-phosphate backbone (isoTBA).

The regioisomeric 3'-deoxy-2'-5'-linked thrombin binding DNA aptamers (isoTBAs) were chemically synthesized and their ability to form unimolecular anti-parallel G-quadruplexes in the presence of K(+) ions was evaluated. These modified sequences retain the function of the native thrombin binding aptamer (TBA), exhibit better stability against exonuclease and are capable of slowing down the process of blood clotting.

C3'-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: novel ethano locked PNA (ethano-PNA).

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60-80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated.

Detection and knockdown of microRNA-34a using thioacetamido nucleic acid.

Thioacetamido nucleic acids (TANA) contain a backbone modification of dinucleotides consisting of a 5-atom amide linker N3'-COCH2-S-CH2 at thymidine or thymidine-cytidine dimer blocks. Here, the chemical synthesis of the TANA linked 5-methyl-cytidine-cytidine ((Me)cc) dimer block and its incorporation into the DNA sequence, complementary to human microRNA 34 (miR-34) is described. Further, for the first time, we demonstrate the biological applications of TANA modified oligonucleotides in detection and intracellular knockdown of a cancer related microRNA in comparison with DNA containing locked nucleic acid (LNA) and 2'-O-methyl modifications.

Synthesis and structural studies of S-type/N-type-locked/frozen nucleoside analogues and their incorporation in RNA-selective, nuclease resistant 2'-5' linked oligonucleotides.

2'-endo locked or frozen (S-type)/3'-endo locked or frozen (N-type) nucleoside analogues were synthesized. Conformational analysis based on (3)J(HH) and NOE measurements is presented which is further confirmed by X-ray crystal structural studies. 2'-5'isoDNA oligonucleotides (ON) were synthesized using these modified nucleoside analogues and UV-T(m) studies of the resultant 2'-5'isoDNA : RNA duplexes reflect the site- and sequence-dependent effects and confirm that the S-type sugar conformations were preferred over the N-type sugar geometry in such duplexes.

Comparing the interactions of DNA, polyamide (PNA) and polycarbamate nucleic acid (PCNA) oligomers with graphene oxide (GO).

The fluorescently labelled short octameric oligothymine sequences of DNA, PNA and PCNA were used in fluorescence quenching studies in conjunction with dispersed graphene oxide. The measurable restoration of their fluorescence by complementary oligodeoxyadenylate was compared. This is the first study aimed at replacing the natural DNA probes with synthetic DNA mimics that show excellent properties in terms of formation of very strong duplexes with cDNA in addition to their stability towards proteases and nucleases.

Highly efficient (R-X-R)-type carbamates as molecular transporters for cellular delivery.

The (R-X-R) motif-containing arginine-rich peptides are among the most effective cell-penetrating peptides. The replacement of amide linkages in the (R-X-R) motif by carbamate linkages as in (r-ahx-r)(4) or (r-ahx-r-r-apr-r)(2) increases the efficacy of such oligomers several-fold. Internalization of these oligomers in mammalian cell lines occurs by an energy-independent process.

Design and synthesis of dephosphono DNA analogues containing 1,2,3-triazole linker and their UV-melting studies with DNA/RNA.

This article describes the synthesis of 3'/5' linked 1,2,3-triazolyl dithymidine derivatives, their incorporation into oligonucleotides, and evaluation of their thermal stabilities toward complementary DNA/RNA.

Probing the furanose conformation in the 2'-5'strand of isoDNA : RNA duplexes by freezing the nucleoside conformations.

Sugar conformations in the isoDNA strand of isoDNA : RNA duplexes are preferred S-type locked/frozen in contrast to N-type locked conformations preferred in DNA : RNA duplexes.

Ferrocene-bis(thymine/uracil) conjugates: base pairing directed, spacer dependent self-assembly and supramolecular packing.

X-ray crystallographic studies of methylene linked Ferrocene-bis(thymine/uracil) conjugates Fc(T:T)(M) and Fc(U:U)(M) reveal base dependent 2-D supramolecular assemblies generated via wobble self-pairing for bis-thymine and reverse wobble self-pairing for bis-uracil conjugates, differing in architecture from the corresponding butylene spacer linked conjugates.

Probing binding preferences of DNA and RNA: backbone chirality of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids to differentiate DNA versus RNA selective binding.

Subtle differences in RNA and DNA duplex geometry could be sensed by the changed stereochemistry at 3'-amino function in the 5-atom thioacetamido linker of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids modified oligomers. In contrast to the preferred N-type sugar conformations for either 3'- ribo- or xylo amino nucleosides, predominant S-type sugar conformations were found in the dimers. Although the CD spectral differences for the dimer blocks were found to be identical for those found in phosphodiester linked ribo/xylo dimers, the 5-atom thioactamido linker could reverse the RNA binding selectivity to DNA binding selectivity by the change in configuration at the 3'-amino-substituted sugar.